Figure 3.

Blockage of α₄β₁ paxillin associations interferes with shear resistance developed by wt α₄. (A) JB4 cells expressing either wt α₄ or α₄(Y991A) were pretreated for 15 min with A7B7C7, a cell-permeable inhibitor of paxillin binding to the α₄ tail, or with the control compound A6B6C6, both present at 5 μM. The shear resistance of carrier or compound-treated cells developed after 1-min adhesion to sVCAM-1 (2,220 sites/μm²) was determined as in Fig. 1. Results are mean ± range of two experimental fields. The experiments depicted are each representative of four independent tests. *, P < 0.001 (a two-tailed paired t test) for control compared with A7B7C7-treated cells at 0.5 dyn/cm². (B) JB4 cells expressing wt α₄ were transfected with either paxillin-specific or control luciferase siRNA. Total lysates of each group were immunoblotted with paxillin- or tubulin-specific mAbs. Densitometric analysis reveals a decrease of 70 and 75% in paxillin content in JB4 expressing either wt or α₄(Y991A), respectively. (C) Paxillin silencing impairs resistance to detachment from sVCAM-1 developed by wt α₄β₁ but not α₄(Y991A). The shear resistance of the indicated cells was determined as in A. Results are representative of three independent experiments. Error bars represent SD.